Antibody dependent cellular cytotoxicity (ADCC), mediated by engagement of IgG Fc receptors (Fc3R), has emerged as a critical factor in the in vivo efficacy of anti-tumor antibodies. However, it is still unclear what role individual effector cells play in the anti- tumor effect of these antibodies. In particular, the role of dendritic cells has been brought into focus based on the observations that clinical response is often enhanced in patients re-treated with therapeutic, anti-tumor antibodies. We will investigate this "vaccinal" effect of anti-TRYP1 antibodies in the B16 melanoma model by exploiting the Fc3R conditional knockout strains we have generated. In Aim 1 we will determine the relative contributions of effector cells, such as macrophages, and antigen presenting cells, typified by dendritic cells, to the in vivo tumor modulating activity of cytotoxic IgG antibodies in the B16 melanoma model. IgG1, 2a and 2b antibodies of the TA99 series will be studied in DC and macrophage restricted Fc3RIII and IV knockout strains in both subcutaneous and metastatic B16 tumor models. Protection and treatment models will be explored for both primary and rechallenge studies. Aim 2 will extend on these studies to focus on the role of Fc3R expression on DCs in inducing active immunity to melanoma by inducing anti-tumor immunity by directly targeting DCs with an IgG antibody engineered to deliver a tumor antigen and Fc engineered to induce DC maturation through FcR mediated ITAM signaling. We will engineer the anti-DEC205 antibody with an immunogenic variant of TYRP1 to create DEC-205/ Tyrp1455/522 which will be further modified with an IgG Fc optimized to bind activation FcRs preferentially over the inhibitory FcRs. Animals immunized with this construct will be characterized for antigen presentation, DC maturation and protection from B16 tumor challenge. The adjuvant effect of TA99 antibodies and anti-CTLA4 will be further evaluated in these vaccination studies. PUBLIC HEALTH RELEVANCE: Anti-tumor antibodies have emerged as a significant clinical modality in the treatment of metastatic disease. Improving and extending the activity of these antibodies requires a detailed knowledge of their mechanism of action. The studies proposed will elucidate the relative contributions of macrophages and dendritic cells to the in vivo activity of anti-melanoma antibodies and will explore a novel approach to inducing active immunity to melanoma by directly targeting dendritic cells with a melanosome antigen.